The extrinsic pathway of blood coagulation involves the sequential activation of coagulation factors initiated by tissue factor (TF) leading to thrombin generation. The thrombin initially produced causes platelets to aggregate and generates a fibrin network. The fibrin network stabilizes the platelet aggregate forming the hemostatic plug which maintains the integrity of the circulatory system after vessel wall damage. The deregulation of the anticoagulant pathways, which effectively restrict hemostatic reactions to local vascular damage with responses of appropriate intensity may lead to the unwanted occlusion of blood vessels, i.e., thrombosis.
The procoagulant process which leads to thrombin formation starts with the binding of factor VII(a) to its cofactor tissue factor (TF), an integral membrane protein which is exposed by cellular injury. The factor VIIa.multidot.TF complex converts the zymogens actor X and factor IX into active enzymes by limited proteolysis (1). Activated factor IX (factor IXa) binds to its cofactor, factor VIIIa, after its activation, to form a second complex which activates factor X at a more efficient rate than factor VIIa.multidot.TF. Activated factor X (factor Xa) in complex with activated factor V (factor Va) forms the prothrombinase complex (factor Xa.multidot.factor Va), which converts prothrombin into thrombin. A phospholipid membrane provided by damaged and/or activated cells is essential for the assembly of each of the vitamin K dependent enzyme complexes. [For reviews on blood coagulation and the phospholipid dependent reactions is blood coagulation see respectively references 2 and 3.] Once formed, thrombin accelerates its own generation by positive feedback reactions. Thrombin activates the procofactors factor V and factor VIII by limited proteolysis into their active forms and may also activate factor XI which can accelerate the reaction by factor IX activation (4).
In normal hemostasis the procoagulant system is retarded by anticoagulant mechanisms which serve to localize and attenuate the hemostatic reaction. The anticoagulant systems consist of stoichiometric protease inhibitors and the dynamic protein C pathway. The tissue factor pathway inhibitor (TFPI) is a stoichiometric protease inhibitor which regulates the initiation of coagulation by inhibition of factor VIIa.multidot.TF activity in a factor Xa dependent manner. [For a review on TFPI see reference 5.] TFPI inhibits the proteolytic activity of factor Xa by reversible binding to the active site of factor Xa. Factor Xa is a cofactor for TFPI inhibition of factor VIIa.multidot.TF activity by formation of a quaternary complex factor Xa.multidot.TFPI.multidot.factor VIIa.multidot.TF. Although no deficiency states are known in humans, in vivo the relevance of TFPI was shown in rabbits by the sensitization to TF triggered disseminated intravascular coagulation (DIC) after immunodepletion of TFPI (6). The inhibition was suggested to be primarily the result of inhibition of factor VIIa.multidot.TF since DIC triggered by factor Xa directly was not affected by TFPI (7). At plasma concentrations of TFPI, direct inhibition of prothrombinase activity is only moderate (8) while inhibition of factor VIIa.multidot.TF activity goes to completion in the presence of physiological concentrations of TFPI (9,10) when factor Xa is present.
A dynamic negative feedback mechanism for thrombin activation is provided by the protein C pathway. This pathway is initiated with the binding of thrombin to thrombomodulin, which is constitutively expressed by endothelial cells. [For a review on thrombomodulin and protein C see reference 12]. The thrombin.multidot.thrombomodulin complex activates plasma protein C. Thrombin alone may activate protein C in the absence of calcium ions, however at physiologic concentrations of calcium ions thrombomodulin is required to activate protein C at a significant rate. Recently the first report of an individual suffering from thrombosis associated with a thrombomodulin mutation leading to a defective protein was published (13). The thrombotic events observed in the reported thrombomodulin deficient individual conclusively identifies the significance of thrombomodulin as a natural anticoagulant.
The important role of protein C as an anticoagulant was secured by numerous reports of thrombotic complications in protein C deficient individuals. Activated protein C exerts its inhibitory effect on coagulation by proteolytic inactivation of the procofactors factor V and factor VIII and/or their activated forms, factor Va and factor VIIIa (15-23).
The inactivation of human factor Va occurs largely as a sequential process (26,27). The preferred first cleavage occurs at Arg.sup.506 and is followed by cleavage at Arg.sup.306 and Arg.sup.679. The phospholipid dependent cleavage at Arg.sup.306 is associated with the major loss of cofactor activity (26,27), while cleavage at Arg.sup.506 is only accompanied by a limited loss of activity. A poor anticoagulant response of plasma to activated protein C (24) was recently correlated with a single point mutation in the factor V.sup.LEIDEN gene which leads to an Arg.sup.506.fwdarw.Gin substitution in the factor V molecule (25). Approximately 3-5% of the Caucasian population is heterozygous for this mutation which is associated with .about.20% of the cases of familial thrombosis (25). The observed APC resistance of factor VArg.sup.506.fwdarw.Gln is caused by a slower inactivation by APC of activated factor V.sup.LEIDEN because it lacks the APC cleavage site at Arg.sup.506 (26). The observed "resistances" toward APC inactivation of factor VArg.sup.506.fwdarw.Gln is a consequence of slower cleavage at Arg306 when the 506-507 peptide bond is intact(26). Inactivation of the procofactor factor VArg.sup.506.fwdarw.Gln is not impaired, indicating that the Arg.sup.306 cleavage site is already available in intact factor V and that the increased rate of inactivation of factor Va versus V is the result of a more prominent exposure of Arg.sup.506 after activation of factor V. The kinetics of activation and inactivation of factor V and factor Va are thus of great significance for the observed resistance of factor VaArg.sup.506.fwdarw.Gln (26).
Factor Va in complex with factor Xa has been reported to be protected against inactivation by APC (28). This coupled to the structural similarity of APC and factor Xa and the similarity of interaction of both enzymes to the cofactor suggests that they compete for the same site (29). The rate of inactivation of factor Va is therefore dependent upon the concentration of formed Xa versus the concentration of formed APC.
Protein S is a vitamin K-dependent coagulation factor involved in the regulation of the anticoagulant activity of APC. [For a review on protein S see reference 30]. Protein S is thought to serve as a non-enzymatic cofactor for APC. In plasma, approximately 60% of protein S is completed to C4b-binding protein (C4BP), a component of the complement system and, when bound to C4BP, protein S is unable to function as a cofactor for APC (30). Protein S has been reported to neutralize the protective effect of factor Xa on the APC dependent inactivation of factor Va (31). Because only a 2-fold potentiation by protein S of the inactivation of factor Va by APC in the absence of factor Xa is observed, the deprotection by protein S of factor Va in the presence of factor Xa has been suggested as a possibly important physiological function of protein S. Other suggested functions of protein S include a direct inhibition of prothrombinase activity by protein S, independent of APC (32-34). This effect of protein S was shown to be very significant under conditions of flow and was hypothesized to result for a large part by the occupation of protein S of plant membranes via the high affinity membrane interaction of protein S, which blocks sites of assembly of the vitamin K dependent enzyme complexes. Although at the present time the physiological functions of protein S remains to be clarified, the numerous reports of protein S deficiency associated with familial thrombosis demonstrate the importance of protein S as a coagulation inhibitor (35,36). Rosing et al. (37) conclude in a recent report that protein S normalizes the inactivation of factor Va.sup.LEIDEN by APC, and hypothesize that this normalization by protein S may be the reason why only a relatively small percentage of the individuals with a factor V.sup.LEIDEN mutation develop thrombosis.
Recently an empirical model for the tissue factor pathway to thrombin using purified coagulation factors (38) has been developed as well as a theoretical mathematical model (39) which simulates thrombin formation based upon known kinetic constants for the essential activation reactions thought to be involved in blood coagulation. The empirical model has been expanded to include the stoichiometric inhibitors TFPI and AT-III (40) and the dynamic protein C pathway (41). It was found that thrombin generation initiated by low pM concentrations of factor VIIa.multidot.TF was effectively neutralized by the combination of TFPI and the protein C pathway after the initial generation of a limited amount of thrombin. At physiological concentrations of TFPI and protein C the effectiveness of this combination of inhibitors to inhibit the reaction was shown to be governed by the relative concentrations of thrombomodulin and factor VIIa.multidot.TF. Of interest here is that this combination of inhibitors are both produced by and are present on the endothelium in vivo; thrombomodulin as a transmembrane protein constitutively expressed on the luminal side of the endothelial cells (12) and full-length TFPI bound to endothelium exposed heparan sulfate-like glycosaminoglycans (42-45).
There is a need in the art for methods to identify those at risk due to a factor V.sup.LEIDEN mutation. There is also a need in the art for prophylactic methods to reduce that risk.